A, Representative scarred and vascularized blebs after glaucoma filtration surgery. B, Mean central bleb area, maximal bleb area, and bleb height grades (1 indicating 0%; 2, 25%; 3, 50%; 4, 75%; and 5, 100%). Mean bleb vascularity was graded on a scale in which 1 indicates avascularity; 2, normal; 3, mild; 4, moderate; and 5, severe hyperemia. Error bars indicate SEM.
A-D, Data are means. Error bars indicate SEM. Snellen equivalents are 20/20, 20/32, 20/50, 20/80, 20/125, and 20/200. E, Percentage of patients receiving different types of antiglaucoma treatment. C/D indicates cup-disc; IOP, intraocular pressure; and VA, visual acuity.
IL6 (A) and PRG4 (B) genes were measured by real-time quantitative polymerase chain reaction. All mean messenger RNA values were normalized relative to that of GAPDH, and triplicate experiments were performed for each condition. Error bars indicate SEM. Spearman correlation is shown between the IL6 gene and number of glaucoma operations (C) and the IL6 gene and logMAR visual acuity (Snellen equivalents given in Figure 1) (D). Spearman correlation is shown between the PRG4 gene and number of glaucoma operations (E) and the PRG4 gene and logMAR visual acuity (F). The values in more than 1 cell line were the same for PRG4 gene expression. FF indicates fibrotic fibroblast; NF, nonfibrotic fibroblast; and VA, visual acuity.
Sections of fibrotic and nonfibrotic conjunctiva tissues were stained for the IL6 (A) and PRG4 (B) proteins. Arrowheads indicate areas of high IL6 and PRG4 staining (original magnification ×20 [upper images] and ×40 [lower images]). Scale bar = 50 μm.
Fibrotic fibroblasts and nonfibrotic fibroblasts were seeded onto coverslips and stained for IL6 (A) and PRG4 (B). Scale bar = 30 μm. Mean fluorescent intensity of IL6 (C) and PRG4 (D) staining were calculated using a bespoke image processing algorithm written in MATLAB. Three randomly chosen fields were imaged per condition. Error bars indicate SEM.
eTable 1. Patient Demographics
eTable 2. Fibrotic and Nonfibrotic Fibroblast Cell Lines
eTable 3. Correlation of IL6 and PRG4 Gene Expression With Clinical Phenotype
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Yu-Wai-Man C, Tagalakis AD, Meng J, et al. Genotype-Phenotype Associations of IL6 and PRG4 With Conjunctival Fibrosis After Glaucoma Surgery. JAMA Ophthalmol. 2017;135(11):1147–1155. doi:10.1001/jamaophthalmol.2017.3407
Are IL6 and PRG4 potential novel tissue biomarkers of conjunctival fibrosis after glaucoma surgery?
In this cross-sectional study, IL6 expression was upregulated and PRG4 expression was downregulated in fibrotic conjunctiva, and both correlated strongly with the number of glaucoma operations and logMAR visual acuity.
These results support IL6 and PRG4 as potential tissue biomarkers of disease severity and prognosis in conjunctival fibrosis after glaucoma surgery; longitudinal studies are needed to validate these biomarkers of fibrosis in the future.
Postsurgical fibrosis is a critical determinant of the long-term success of glaucoma surgery, but no reliable biomarkers are currently available to stratify the risk of scarring.
To compare the clinical phenotype of patients with conjunctival fibrosis after glaucoma surgery with candidate gene expression tissue biomarkers of fibrosis.
Design, Setting, and Participants
In this cross-sectional study, 42 patients were recruited at the time of glaucoma surgery at the Moorfields Eye Hospital from September 1, 2014, to September 1, 2016. The participants were divided into those with fibrosis and those without fibrosis.
Main Outcomes and Measures
Genotype-phenotype correlations of the IL6 or PRG4 gene and detailed clinical phenotype. The IL6 and PRG4 protein expression in conjunctival tissues was also assessed using in situ immunohistochemical analysis. Central bleb area, maximal bleb area, and bleb height were graded on a scale of 1 to 5 (1 indicating 0%; 2, 25%; 3, 50%; 4, 75%; and 5, 100%). Bleb vascularity was graded on a scale of 1 to 5 (1 indicating avascularity; 2, normal; 3, mild; 4, moderate; and 5, severe hyperemia).
A total of 42 patients were recruited during the study period; 28 participants (67%) had previously undergone glaucoma surgery (fibrotic group) (mean [SD] age, 43.8 [3.6 years]; 16 [57%] female; 22 [79%] white), and 14 participants (33%) had not previously undergone glaucoma surgery (nonfibrotic group) (mean [SD] age, 47.7 [6.9] years; 4 [29%] female; 9 [64%] white). The fibrotic group had marked bleb scarring and vascularization and worse logMAR visual acuity. The mean (SD) grades were 1.4 (0.1) for central bleb area, 1.4 (0.1) for bleb height, and 3.4 (0.2) for bleb vascularity. The IL6 gene was upregulated in fibrotic cell lines (mean, 0.040) compared with nonfibrotic cell lines (mean, 0.011) (difference, 0.029; 95% CI, 0.015-0.043; P = .003). The PRG4 gene was also downregulated in fibrotic cell lines (0.002) compared with nonfibrotic cell lines (mean, 0.109; difference, 0.107; 95% CI, 0.104-0.110; P = .03). The study found a strong correlation between the IL6 gene and the number of glaucoma operations (r = 0.94, P < .001) and logMAR visual acuity (r = 0.64, P = .03). A moderate correlation was found between the PRG4 gene and the number of glaucoma operations (r = −0.72, P = .005) and logMAR visual acuity (r = −0.62, P = .03).
Conclusions and Relevance
IL6 and PRG4 represent potential novel tissue biomarkers of disease severity and prognosis in conjunctival fibrosis after glaucoma surgery. Future longitudinal studies with multiple postoperative measures are needed to validate the effect of these potential biomarkers of fibrosis.
Fetal skin wound healing is scarless and fundamentally different from adult wound healing.1,2 Scarless fetal wound healing is characterized by little inflammation, minimal fibroblast proliferation, and decreased levels of potent inflammatory cytokines, such as interleukin 6 (IL-6).3 Interleukin 6 controls the effector characteristics of various T-cell subsets, including helper T17 cells, helper T22 cells, and certain IL-10–secreting subsets.4 Interleukin 6 also plays an important role in pulmonary fibrosis,5 peritoneal fibrosis,6 renal interstitial fibrosis,7 and cancer-associated fibroblasts.8
Scarless fetal wound healing is associated with high levels of hyaluronic acid,9 which increases the expression of proteoglycan 4 (PRG4).10 Also known as lubricin, PRG4 is a lubricating mucinlike glycoprotein that has been detected at the ocular surface.11 It is downregulated in palmar fascia fibroblasts from patients with Dupuytren contracture.12 Moreover, increasing PRG4 expression using recombinant human PRG4 treatment decreases α-smooth muscle actin expression in lens epithelial cells activated with transforming growth factor β2 and may prevent the full myofibroblast phenotype in posterior capsular opacification.13
Detailed clinical phenotyping and effective biobanking of large patient cohorts are critical to the study of putative biomarkers of disease severity and prognosis in ocular fibrosis.14 Afro-Caribbean patients have a genetic predisposition to ocular and keloid scarring.15 Dupuytren contracture is also a familial disorder that is highly prevalent in individuals of Northern European descent.16 However, there is a current lack of reliable biomarkers to stratify the risk of scarring in the eye. The ability to determine patients’ risk of scarring and to tailor the antifibrotic treatment regimen to each patient would be clinically useful to prevent undertreating or exposing them to unnecessary treatments with potential adverse effects. In this study, we sought to determine whether correlation of the clinical phenotype of patients with conjunctival fibrosis with altered gene expression revealed IL6 (OMIM 147620) and PRG4 (OMIM 604283) as potential novel tissue biomarkers of fibrosis.
We recruited patients with glaucoma at the time of surgery at the Moorfields Eye Hospital, London, England, from September 1, 2014, to September 1, 2016. All participants gave written informed consent after an explanation of the nature and possible consequences of the study. All data were anonymized and deidentified. The inclusion criteria were age older than 18 years and a plan to undergo glaucoma tube surgery. The exclusion criterion was previous conjunctival surgery other than glaucoma surgery. The study was performed according to approval of the National Research Ethics Service Committee London-Dulwich and the tenets of the Declaration of Helsinki.17
The participants were divided into 2 groups: participants who had previous glaucoma surgery (fibrotic group) and participants with no previous glaucoma surgery (nonfibrotic group). All study participants underwent a standardized ophthalmic examination before surgery, including best-corrected visual acuity, intraocular pressure (IOP), lens status, and cup-disc ratio. We also collected detailed information on patient demographics, including age, sex, ethnicity, and type of glaucoma.
We assessed each study participant with previous glaucoma surgery using the Moorfields bleb grading system.18 Central bleb area, maximal bleb area, and bleb height were graded on a scale of 1 to 5 (1 indicating 0%; 2, 25%; 3, 50%; 4, 75%; and 5, 100%). Bleb vascularity was graded on a scale of 1 to 5 (1 indicating avascularity; 2, normal; 3, mild; 4, moderate; and 5, severe hyperemia).
We collected information on the number of antiglaucoma eyedrops, including β-blockers, prostaglandin analogues, carbonic anhydrase inhibitors, α-agonists, and pilocarpine, as well as the number of patients taking oral acetazolamide. We also recorded any previous surgical and laser treatments, including the number of trabeculectomies, glaucoma tube operations, and diode laser treatments.
We established fibrotic fibroblast (FF) and non–fibrotic fibroblast (NF) primary cell lines from conjunctival tissues collected from study participants in the fibrotic group and study participants in the nonfibrotic group, respectively. The conjunctival tissues were mechanically dispersed, and the tissue fragments were placed in tissue culture dishes with Dulbecco modified Eagle medium (Invitrogen), 10% fetal calf serum, 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 2mM of l-glutamine at 37°C with 5% carbon dioxide.19 After outgrowth from the explant, the fibroblasts were trypsinized and cultured routinely in the aforementioned medium. Fibroblast cell lines in early passages 1 to 2 were used in the experiments.
Real-time quantitative polymerase chain reactions (PCRs) were performed using a Platinum quantitative PCR master mix (Thermo Fisher Scientific) on a CFX Real-Time PCR detection system (Bio-Rad). The Taqman gene expression assays were IL6 (Hs00985639_m1), PRG4 (Hs00981633_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (OMIM 138400) (Hs02758991_g1) (Thermo Fisher Scientific). All messenger RNA values were normalized relative to that of GAPDH, and triplicate experiments were performed for each condition.
Fibrotic and nonfibrotic human conjunctival tissues were fixed in formalin for 24 hours. The samples were dehydrated through an alcohol gradient (70%, 95%, and 100% Industrial Methylated Spirit) followed by chloroform clearance on a Peloris II tissue processing machine (Leica Camera AG). The samples were embedded in paraffin wax using a Tissue-Tek TEC embedding machine (Sakura International Inc). Tissue sections were cut on a manual rotary microtome (HM340E, Thermo Fisher Scientific) at a section thickness of 3 µm using S35 microtomy blades (FEATHER Safety Razor Co Ltd). The sections were mounted on slides (Xtra Adhesive, Leica Camera AG), air dried for 60 minutes, and heated for 60 minutes at 70°C before staining. The sections were dewaxed and stained on a Bond-Max automated immunostainer (Leica Camera AG). The sections were incubated with IL-6 (1:400, rabbit polyclonal ab6672, Abcam) or PRG4 (1:200, polyclonal rabbit HPA028523, Atlas) antibody for 30 minutes at room temperature.
The FF and NF cell lines were seeded onto coverslips at 1 × 105 cells per well in 6-well plates. The cells were fixed with 4% paraformaldehyde for 15 minutes and incubated with blocking solution (phosphate-buffered saline that contained 10% normal goat serum and 0.03% Triton X-100) for 30 minutes. The cells were then incubated with primary antibodies against IL-6 (1:100, ab6672, Abcam) or PRG4 (1:100, HPA028523, Atlas) for 2 hours followed by Alexa 488–conjugated goat antirabbit IgG (H+L) (1:500, Thermo Fisher Scientific) for 1 hour. The coverslips were then rinsed with phosphate-buffered saline and mounted onto slides with mounting medium (Dako) that contained 10 μg/mL of 4',6-diamidino-2-phenylindole. Three randomly chosen fields per sample were imaged at ×20 magnification using a DM4000B microscope (Leica Camera AG). Stained areas of IL-6 and PRG4 were calculated using a bespoke image processing algorithm written in MATLAB. Each image was first converted into a grayscale image ranging from a level of 0 for black to 255 for white. The maximum background grayscale level of each image was then identified, and any grayscale value above it was considered as staining.
All data are presented as mean (SEM). Statistical analysis was performed using the unpaired, 2-tailed t test to calculate differences in individual P values. P < .05 (2-sided) was considered to be statistically significant. We performed genotype-phenotype comparisons using the Spearman correlation of the IL6 or PRG4 gene vs bleb area, bleb height, bleb vascularity, number of glaucoma operations, age, IOP, logMAR visual acuity, cup-disc ratio, and number of antiglaucoma eyedrops.
A total of 42 patients were recruited during the study period; 28 participants (67%) had previous glaucoma surgery (fibrotic group), and 14 participants (33%) had no previous glaucoma surgery (nonfibrotic group). Most study participants were white: 22 white (79%), 4 Asian (14%), and 2 Afro-Caribbean (7%) in the fibrotic group and 9 white (64%), 4 Asian (29%), and 1 Afro-Caribbean (7%) in the nonfibrotic group (eTable 1 in the Supplement). The difference in age between groups was not significant, with a mean (SD) age of 43.8 (3.6) years in the fibrotic group and 47.7 (6.9) years in the nonfibrotic group (P = .58). In the fibrotic group, 12 patients (43%) were male and 16 (57%) were female; in the nonfibrotic group, 10 (71%) were male and 4 (29%) were female.
All participants in the fibrotic group had marked bleb scarring and vascularization (Figure 1A). On the basis of the Moorfields bleb grading system, the central bleb area was 1.4 (0.1), maximal bleb area was 1.4 (0.1), bleb height was 1.4 (0.1), and bleb vascularity was 3.4 (0.2) (Figure 1B). The fibrotic participants had multiple glaucoma operations, with a mean of 1.5 (range, 1-3). Among the fibrotic group, 13 participants (47%) had trabeculectomy surgery, 4 participants (14%) had glaucoma tube surgery, and 11 participants (39%) had multiple glaucoma operations (eTable 1 in the Supplement).
The study participants in the fibrotic group had worse best-corrected visual acuity, with a mean logMAR vision of 0.58 (Snellen equivalent, 20/80) compared with 0.31 (Snellen equivalent, 20/40) in the nonfibrotic group (difference, 0.27 [Snellen equivalent, 20/40]; 95% CI, 0.11-0.42 [Snellen equivalent, 20/25-20/50]; P = .04) (Figure 2A). The fibrotic group had a mean preoperative IOP of 22.3 mm Hg, and the nonfibrotic group had a mean preoperative IOP of 27.1 mm Hg (difference, 4.8; 95% CI, 1.8-9.1; P = .13) (Figure 2B). Most participants had advanced disc cupping, with a cup-disc ratio of 0.87 in the fibrotic group and 0.84 in the nonfibrotic group (difference, 0.03; 95% CI, −0.01 to 0.07, P = .35) (Figure 2C). Most study participants were also using multiple antiglaucoma eyedrops, with a mean of 3.6 in the fibrotic group and 3.8 in the nonfibrotic group (difference, 0.2; 95% CI, −0.2 to 0.7; P = .55) (Figure 2D). A high percentage of participants in the fibrotic and nonfibrotic groups were using topical β-blockers, prostaglandin analogues, carbonic anhydrase inhibitors, α-agonists, and oral acetazolamide (Figure 2E).
We next established FF (FF3, FF13, FF14, FF15, FF16, and FF17) and NF (NF1, NF4, NF7, and NF8) primary cell lines from conjunctival tissue samples collected from 10 patients in the study (eTable 2 in the Supplement). We performed real-time quantitative PCR to compare the IL6 and PRG4 genes in the FF and NF cell lines. The IL6 gene was upregulated in the FF cell lines (mean, 0.040) compared with the NF cell lines (mean, 0.011) (difference, 0.029; 95% CI, 0.015-0.043; P = .003). FF14 and FF16 had the highest IL6 gene expression, whereas NF4 and NF7 had the lowest IL6 gene expression (Figure 3A). The PRG4 gene was also downregulated in the FF cell lines (0.002) compared with the NF cell lines (mean, 0.109; difference, 0.107; 95% CI, 0.104-0.110; P = .03). NF1 and NF4 had the highest PRG4 gene expression, whereas FF13 and FF15 had the lowest PRG4 gene expression (Figure 3B).
We also performed Spearman correlation between the IL6 or PRG4 gene and the detailed clinical phenotype of patients. The Spearman correlation coefficient (r) ranges in value from −1 to +1. The larger the absolute value of the coefficient, the stronger the correlation between the variables. A positive r indicates a positive association between the 2 variables, whereas a negative r indicates a negative association. We found a strong correlation between the IL6 gene and the number of glaucoma operations (r = 0.94, P < .001) (Figure 3C) and logMAR visual acuity (r = 0.64, P = .03) (Figure 3D). A moderate correlation was also found between the PRG4 gene and the number of glaucoma operations (r = −0.72, P = .005) (Figure 3E) and logMAR visual acuity (r = −0.62, P = .03) (Figure 3F). However, we found no correlations between the IL6 or PRG4 gene and the central bleb area, maximal bleb area, bleb height, and bleb vascularity (eTable 3 in the Supplement). In addition, no correlations were found between the IL6 or PRG4 gene and patient age, IOP, cup-disc ratio, and number of antiglaucoma eyedrops (eTable 3 in the Supplement).
We further stained fibrotic and nonfibrotic conjunctival tissues from the glaucoma study participants for IL6 and PRG4 protein expression. Fibrotic conjunctival tissues after glaucoma surgery undergo marked histopathologic changes compared with nonfibrotic conjunctival tissues. Similar to the gene expression patterns, fibrotic conjunctival tissues expressed increased IL6 and decreased PRG4 protein staining compared with nonfibrotic conjunctival tissues (Figure 4).
In addition, we stained the FF and NF cell lines to compare the distribution of the IL6 and PRG4 proteins in fibrotic and nonfibrotic conjunctival fibroblasts. The FF cell lines had a higher proliferative rate than the NF cell lines, but overall, no differences were found in cell morphologic findings between the 2 groups. There was a mixture of nuclear and cytoplasmic IL6 and PRG4 staining pattern in the FF and NF cell lines (Figure 5A and B). The FF cell lines showed increased IL6 staining compared with the NF cell lines. FF14 and FF16 had the highest IL6 staining, whereas NF1 and NF4 had the lowest IL6 staining (Figure 5C). We, however, did not find any differences in PRG4 staining between the FF and NF cell lines (Figure 5D). This finding could potentially be attributable to the sensitivity and specificity of the PRG4 antibody used in the study.
Deep clinical phenotyping and the use of specific tissue biomarkers represent key aspects of personalized medicine in ocular fibrosis.14 A previous study20 found that there is a distinct fibrosis gene signature in the conjunctiva after glaucoma surgery, and the RNA signature revealed an upregulation of the IL6 gene and a downregulation of the PRG4 gene in fibrotic human conjunctival fibroblasts. Using detailed genotype-phenotype comparisons, we found that there is a correlation between the IL6 or PRG4 gene with the number of glaucoma operations and logMAR visual acuity. Fibrotic conjunctival tissues after glaucoma surgery also expressed increased IL6 and decreased PRG4 protein staining compared with nonfibrotic conjunctival tissues.
Our results are consistent with key mechanistic pathways in conjunctival fibrosis, namely, extracellular matrix remodeling and the inflammatory response. The extracellular matrix component PRG4 is a serum response factor target gene.21 Serum response factor is a master regulator of cytoskeletal gene expression,21,22 and the myocardin-related transcription factor/serum response factor pathway has been linked to ocular,23-25 vascular,26 skin,27 and lung fibrosis.28 Proteoglycan 4 also binds to Toll-like receptors and plays an anti-inflammatory role in downstream signaling pathways, such as IL-6.29-31 Multiple Toll-like receptors can initiate an IL-6 transcriptional response.32 Interleukin 6 is a potent inflammatory cytokine, and signal transduction involves the activation of Jak family members, leading to the activation of transcription factors of the STAT family.33 Another major signaling pathway for IL-6 is the Ras/MAPK and ERK/MAPK cascade.33
Inflammation is a risk factor for scarring after trabeculectomy surgery, and increased inflammatory cells in the conjunctival tissues of patients who previously underwent glaucoma surgery is associated with an increased risk of conjunctival scarring.34,35 Afro-Caribbean patients also represent a high-risk group for conjunctival scarring, and the increased number of conjunctival macrophages may partially explain the tendency for a lower success rate of filtration surgery in this group of patients.36 In addition, long-term treatment with multiple antiglaucoma eyedrops has been identified as a significant risk factor for failure in trabeculectomy because it causes subclinical conjunctival inflammation with increased macrophages and lymphocytes in the conjunctival epithelium.37,38
Higher IL6 levels also correlated strongly with multiple failed glaucoma operations and worse visual acuities in patients with glaucoma. Trachoma is another conjunctival scarring disease, and IL6 is overexpressed in scarring trachoma fibroblasts.39 Moreover, IL6 is an important immune mediator, and a genome-wide association study further suggests that the genetic associations with trachoma scarring might be focused on processes related to the immune system.40 Similarly, increased IL-6 has been found in dermal interstitial blister fluid from patients with systemic sclerosis.41 High IL-6 expression in patients with early diffuse cutaneous systemic sclerosis is also associated with more severe skin involvement at 3 years and worse long-term survival than in those without elevated IL-6 levels.42
In this study, we found a downregulation of the PRG4 gene in fibrotic human conjunctival fibroblasts after glaucoma filtration surgery. Lower PRG4 levels correlated moderately with multiple failed glaucoma operations and worse visual acuities in patients. Proteoglycan 4 is an important biological modifier that regulates processes such as tissue development, homeostasis, inflammation, innate immune response, and wound healing.43,44 Chronic obstructive pulmonary disease is characterized by a progressive loss of lung function that is caused by repeated pulmonary inflammation.45 Another study46 found that serum PRG4 correlated strongly with the 1-year change in predicted lung forced vital capacity and is a diagnostic biomarker in patients with chronic obstructive pulmonary disease.
New biomarkers of fibrosis may provide sensitive and reproducible means of targeting and personalizing therapy and assessing disease response in the future. Pirfenidone treatment decreased IL-6 levels47 and reduced disease progression in patients with idiopathic pulmonary fibrosis.48 Tocilizumab, a monoclonal antibody against IL-6, did not produce a significant reduction in skin thickening, but there was evidence of reduced decline in lung forced vital capacity in a phase 2 clinical trial in patients with systemic sclerosis.49
Because this was a cross-sectional study, it was not possible to determine whether the observed differences in IL6 and PRG4 expression in FF and NF cell lines preceded fibrosis rather than being the consequence of developing fibrosis. A future longitudinal study of patients undergoing surgery for the first time with multiple postoperative measures of tissue biomarkers will be necessary to validate the effect desired for clinically useful biomarkers. Another limitation is the relatively small sample size, and we are establishing a fibrosis biobank of tissues to validate our results in larger longitudinal studies in the future.
This cross-sectional study found that IL6 is upregulated and PRG4 is downregulated in fibrotic human conjunctiva after glaucoma filtration surgery, and both correlated with the number of glaucoma operations and logMAR visual acuity. Confirmation from future longitudinal studies is needed to have greater confidence that IL6 and PRG4 are potential novel tissue biomarkers of disease severity and prognosis in conjunctival fibrosis after glaucoma surgery.
Corresponding Author: Cynthia Yu-Wai-Man, PhD, FRCOphth, National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital National Health Service Foundation Trust and University College London Institute of Ophthalmology, 11-43 Bath St, London EC1V 9EL, England (email@example.com).
Accepted for Publication: July 27, 2017.
Published Online: September 21, 2017. doi:10.1001/jamaophthalmol.2017.3407
Author Contributions: Dr Yu-Wai-Man had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Yu-Wai-Man.
Acquisition, analysis, and/or interpretation of data: Yu-Wai-Man, Tagalakis, Meng, Bouremel, Lee, Virasami, Hart.
Drafting of the manuscript: Yu-Wai-Man.
Creation of figure illustrations: Yu-Wai-Man, Bouremel, Lee.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Yu-Wai-Man, Bouremel, Lee.
Obtained funding: Yu-Wai-Man, Khaw.
Administrative, technical, or material support: Yu-Wai-Man, Tagalakis, Meng.
Study supervision: Yu-Wai-Man, Khaw.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.
Funding/Support: This research was supported by the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital National Health Service Foundation Trust and University College London Institute of Ophthalmology, the Medical Research Council, and Moorfields Eye Charity.
Role of the Funder/Sponsor: The funding sources had no role in the design or conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
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